The focus of this proposal is on the structure of PARK14-encoded phospholipase A2g6 (PLA2g6), and its unique Ca2+ signaling function, which we have recently discovered to be intimately involved in the genesis of Parkinson's disease (PD). We found that the patients with idiopathic or familial PD associated with specific PARK14 mutations have normal catalytic activity of PLA2g6, but significant deficiency in its store-dependent activation, which results in impairment of Ca2+ entry, depletion of intracellular Ca2+ stores, autophagic dysfunction and other pathological events leading to age-dependent PD. Only full length PLA2g6(L), but not (S) splice variant can restore Ca2+ signaling and prevent PD-associated deficits. Thus, PARK14-encoded PLA2g6(L) emerged as a potential target for PD drug discovery, but the progress is currently halted by the lack of atomic structure of PLA2g6, poor understanding of the structural and functional mechanisms underlying its PD-associated dysfunction, and the fundamental differences between its (L) and (S) variants. Our working hypothesis is that functional PLA2g6(L) represents a tetramer with unique atomic structure that allows this protein to associate with plasma membrane, which is essential for its specific store-operated Ca2+ signaling function and role in human PD. Our multidisciplinary research team will use synergistic approach integrating electron cryomicroscopy (CryoEM), biochemical, molecular, and cellular approaches to identify structural determinants of Ca2+ signaling function and PD-related dysfunction of PLA2g6. Aims are: Aim 1. Determine the 3D structure of full-length PLA2g6(L) and compare it with PLA2g6(S). We will use high-throughput single-particle CryoEM to solve the structure of human recombinant PLA2g6(L) and (S) proteins heterologously expressed and purified from human cells. Aim 2. Investigate structure-based functional properties of PLA2g6(L). Molecular, biochemical and live cell functional assays will be used to validate PLA2g6(L) structure, and confirm some of its functional predictions. We will use mutagenesis and specific peptides to investigate the role of PIN domain for PLA2g6(L) association with plasma membrane and its store-operated Ca2+ signaling function. Expected outcome and impact of the proposed research: We expect to solve the structure of PARK14- encoded full length PLA2g6(L) at atomic (< 3 ) resolution and delineate molecular and structural determinants of its unique Ca2+ signaling function. This multidisciplinary research direction breaks the barriers between basic science and translational medicine, creates a new powerful interface between structural analysis and mechanisms of neurodegeneration, and increases the probability of the major scientific discoveries that can transform multiple fields. Solving the PLA2g6 structure and understanding the mechanism of its PD-associated dysfunction will open doors for development of PLA2g6 targeted therapeutic interventions for human PD.